Anesthetic agents alter the function of the cardiovascular system, resulting in hypotension and peripheral vessel dilatation. Studies have attributed this action to a direct depressant effect on vascular smooth muscle cells, possibly by an impairment of the Ca2+ second messenger system. No conclusive biochemical studies, however, have been done on a cellular level. Endothelial cells may also be involved in this effect, since endothelial cells have been shown to regulate the tonic state of the vessel. Anesthetic agents attenuate the contractile response of both large blood vessels and microvessels to vasoactive substances. Recently, it has been reported that some vasoactive substances act in vascular smooth muscle and in endothelial cells through the phosphatidylinositol (PI) pathway (located in the cell plasma membrane), coupled to an internal Ca2+ signal. Our preliminary studies on the effects of anesthetics on smooth muscle cells show that the barbiturate, pentobarbital (Pb), dose-dependently inhibits PI hydrolysis without affecting cell viability. Pb selectively inhibits PI hydrolysis on what appears to be one of two components of PI hydrolysis (a high affinity component, Km=2.0 nM). This effect with Pb has also been confirmed in an intact vessel. These results suggest that specific changes can be made in the PI pathway of vascular cells, by anesthetic agents. The proposed studies extend our investigations of the effects of anesthetic agents on the PI system in vascular cells to volatile anesthetics such as halothane and isoflurane. We will employ cultured rat smooth muscle and endothelial cells as well as cerebral microvascular endothelial cells in these studies. Techniques will include biochemical assays, radiolabeled assays, and high pressure liquid chromatography (HPLC) to assay for PI and components of the PI pathway and to obtain evidence of a structure-activity relationship with anesthetics. Radioimmunoassays will be used in control studies for C'AMP and C'GMP. We will also measure intracellular Ca2+ levels by spectrofluorometric assay with fluorescent Ca2+ probes (Quin 2/AME and Fura 2), we will correlate the results of PI metabolism and intracellular Ca2+ studies with cell contraction, by use of a specialized microscope and camera system for counting individual cell contractions.